Alzheimer's disease is a progressive neurodegenerative disease causing cognitive decline and memory loss that effects 1 in 8 Americans over the age of 65 (1). It is estimated that by the year 2050 between 11 million and 16 million Americans age 65 and older will be affected (2). Currently, there are strategies to temporarily reduce the symptoms of Alzheimer's, but there are no therapies to stop the progression of neurodegeneration or cure AD (3). There are two main pathophysiological features of AD, beta amyloid plaques and neurofibrillary tangles (4). Neurofibrillary tangles are composed of hyperphosphorylated tau protein (5, 6). Normal tau is a microtubule-associated protein that stabilizes microtubules (7). Hyperphosphorylation of tau decreases its affinity for microtubules and hyperphosphorylated tau sequesters normal tau preventing it from binding to microtubules (8-10). Tau is phosphorylated by numerous kinases, but PP2A has been implicated as being the predominant phosphatase involved in tau dephosphorylation (11-13). Alpha4 has been shown to regulate PP2A stability and expression (14, 15) and localizes to microtubules due to its association with Mid1 (16). Specifically, alpha4 regulates ubiquitination of PP2Ac and its degradation by the proteasome by suppressing polyubiquitination of PP2Ac (15). Based on previous studies and preliminary structures, the working hypothesis is that the UIM of alpha4 binds to monoubiquitinated PP2Ac capping the ubiquitin chain and preventing polyubiquitination and that this protection is regulated through the ubiquitination of alpha4. In order to understand the mechanism by which alpha4 regulates PP2Ac, double electron-electron resonance (DEER) studies will be conducted to look at the role of flexibility of alpha4 in interacting with both PP2Ac and ubiquitin. To investigate the mechanism of alpha4 regulation of PP2A, a neuronal cell line will be transfected with alpha4 constructs that disrupt protein-protein interactions and PP2Ac ubiquitination and stability will be assessed. Activity of PP2A towards tau will be assessed using by measuring levels of phosho-tau using available phospho-tau antibodies. Our data indicate that alpha4 may be regulated via ubiqutination. To investigate this, 293FT cells will be transfected with alpha4 constructs that disrupt protein-protein interactions and alpha4 ubiquitination will be measured. The goal of this project is to elucidate the mechanism of alpha4 regulation of PP2A and investigate the role alpha4 plays in regulating PP2A activity towards tau.